Classically, progesterone is a reproductive hormone functioning via nuclear receptors (PR-B and A) to control gene transcription. Progesterone actions vary in tissues such as an antiproliferative role in endometrium versus a proliferative role in breast epithelial cells. Additionally, immediate nongenomic actions of progesterone have been described in several tissues. Our long-term objective is to identify unique progesterone receptors and signaling pathways that may explain these various actions. Previously, we have identified a novel truncated progesterone receptor named PR-M. Preliminary data suggest that PR-M is located in the mitochondria and modulates mitochondrial calcium entry. We hypothesize that progesterone via PR-M regulates cellular respiration thus affecting metabolic rate. The specific aims are: 1) to localize PR-M to the outer versus inner membrane of the mitochondria; 2) to demonstrate modulation of mitochondrial calcium levels and depolarization by progesterone via PR-M; and 3) to show regulation of cellular ATP production and induction of apoptosis by progesterone via PR-M. These aims will utilize cells transfected to over-express PR-M and breast cancer cells with endogenous expression. Techniques include digitonin subfractionation and immuno-EM [electron microscopy] for No.1, calcium imaging with and epifluorescent microscopy for No. 2 and fluorescent-activated cell sorting for No. 3. A direct mechanism whereby progesterone regulates cellular respiration may affect tissue growth and metabolism during pregnancy and in pathologic conditions such as breast cancer. Relevance to public health: Clinical studies have shown an interaction of progesterone with many disease processes including breast cancer, meningiomas, endometriosis, fibroid uterine tumors and complications of pregnancy such as pre-term labor and preeclampsia. It is now evident that the actions of this hormone are more complex than initially considered and that progesterone works in other ways than controlling the production of proteins. The possibility that progesterone directly affects the metabolism of the cell by altering the activity of the energy producing mitochondria represents a novel action of sex hormones. This could also explain conflicting laboratory observations in which progesterone causes both growth and inhibition of growth of breast cancers. There also exists the possibility that other hormones may affect metabolism in a similar manner. [unreadable] [unreadable] [unreadable]